Branched Acetylene-Containing Poly(Alkylene Oxides, Oxyethylated Polyols or Olefinic Alcohols)

ABSTRACT

The invention provides water-soluble compounds that include a polymer and at least one terminal azide or acetylene moiety. Also provided are highly efficient methods for the selective modification of proteins with PEG derivatives, which involves the selective incorporation of non-genetically encoded amino acids, e.g., those amino acids containing an azide or acetylene moiety, into proteins in response to a selector codon and the subsequent modification of those amino acids with a suitably reactive PEG derivative.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalApplication No. 60/510,169 entitled POLYMER DERIVATIVES which isincorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to derivatives of hydrophilic compounds andpolymers that contain azide or alkyne moieties, methods for theirsynthesis, and methods for their use in modifying the characteristicsand properties of molecules.

2. Background

Covalent attachment of the hydrophilic polymer poly(ethylene glycol),abbreviated PEG, is a method of increasing water solubility andbioavailability and extending the circulation time of many biologicallyactive molecules, including proteins, peptides, and particularlyhydrophobic molecules. PEG has been used extensively in pharmaceuticals,on artificial implants, and in other applications wherebiocompatibility, lack of toxicity, and lack of immunogenicity are ofimportance. In order to maximize the desired properties of PEG, thetotal molecular weight and hydration state of the PEG polymer orpolymers attached to the biologically active molecule must besufficiently high to impart the advantageous characteristics typicallyassociated with PEG polymer attachment, such as increased watersolubility and circulating half life, while not adversely impacting thebioactivity of the parent molecule.

PEG derivatives are frequently linked to biologically active moleculesthrough reactive chemical functionalities, such as lysine, cysteine andhistidine residues, the N-terminus and carbohydrate moieties. Proteinsand other molecules often have a limited number of reactive sitesavailable for polymer attachment. Often, the sites most suitable formodification via polymer attachment play a significant role in receptorbinding, and are necessary for retention of the biological activity ofthe molecule. As a result, indiscriminate attachment of polymer chainsto such reactive sites on a biologically active molecule often leads toa significant reduction or even total loss of biological activity of thepolymer-modified molecule. R. Clark et al., (1996), J. Biol. Chem.,271:21969-21977. To form conjugates having sufficient polymer molecularweight for imparting the desired advantages to a target molecule, priorart approaches have typically involved random attachment of numerouspolymer arms to the molecule, thereby increasing the risk of a reductionor even total loss in bioactivity of the parent molecule.

Reactive sites that form the loci for attachment of PEG derivatives toproteins are dictated by the protein's structure. Proteins, includingenzymes, are built of various sequences of alpha-amino acids, which havethe general structure H₂N—CHR—COOH. The alpha amino moiety (H₂N—) of oneamino acid joins to the carboxyl moiety (—COOH) of an adjacent aminoacid to form amide linkages, which can be represented as—(NH—CHR—CO)_(n)—, where the subscript “n” can equal hundreds orthousands. The fragment represented by R can contain reactive sites forprotein biological activity and for attachment of PEG derivatives.

For example, in the case of the amino acid lysine, there exists an —NH₂moiety in the epsilon position as well as in the alpha position. Theepsilon —NH₂ is free for reaction under conditions of basic pH. Much ofthe art in the field of protein derivatization with PEG has beendirected to developing PEG derivatives for attachment to the epsilon—NH₂ moiety of lysine residues present in proteins. “Polyethylene Glycoland Derivatives for Advanced PEGylation”, Nektar Molecular EngineeringCatalog, 2003, pp. 1-17. These PEG derivatives all have the commonlimitation, however, that they cannot be installed selectively among theoften numerous lysine residues present on the surfaces of proteins. Thiscan be a significant limitation in instances where a lysine residue isimportant to protein activity, existing in an enzyme active site forexample, or in cases where a lysine residue plays a role in mediatingthe interaction of the protein with other biological molecules, as inthe case of receptor binding sites.

A second and equally important complication of existing methods forprotein PEGylation is that the PEG derivatives can undergo undesiredside reactions with residues other than those desired. Histidinecontains a reactive imino moiety, represented structurally as —N(H)—,but many derivatives that react with epsilon —NH₂ can also react with—N(H)—. Similarly, the side chain of the amino acid cysteine bears afree sulfhydryl group, represented structurally as —SH. In someinstances, the PEG derivatives directed at the epsilon —N(H)— group oflysine also react with cysteine, histidine or other residues. This cancreate complex mixtures of PEG-derivatized bioactive molecules and risksdestroying the activity of the bioactive molecule being targeted. Itwould be desirable to develop PEG derivatives that permit a chemicalfunctional group to be introduced at a single site within the proteinthat would then enable the selective coupling of one or more PEGpolymers to the bioactive molecule at specific sites on the proteinsurface that are both well-defined and predictable.

In addition to lysine residues, considerable effort in the art has beendirected toward the development of activated PEG reagents that targetother amino acid side chains, including cysteine, histidine and theN-terminus. U.S. Pat. No. 6,610,281. “Polyethylene Glycol andDerivatives for Advanced PEGylation”, Nektar Molecular EngineeringCatalog, 2003, pp. 1-17. Cysteine residue can be introducedsite-selectively into the structure of proteins using site-directedmutagenesis and other techniques known in the art, and the resultingfree sulfhydryl moiety can be reacted with PEG derivatives that bearthiol-reactive functional groups. This approach is complicated, however,in that the introduction of a free sulfhydryl group can complicate theexpression, folding and stability of the resulting protein. Thus, itwould be desirable to have a means to introduce a chemical functionalgroup into bioactive molecules that enables the selective coupling ofone or more PEG polymers to the protein while simultaneously beingcompatible with (i.e., not engaging in undesired side reactions with)sulfhydryls and other chemical functional groups typically found inproteins.

As can be seen from a sampling of the art, many of these derivativesthat have been developed for attachment to the side chains of proteins,in particular, the —NH₂ moiety on the lysine amino acid side chain andthe —SH moiety on the cysteine side chain, have proven problematic intheir synthesis and use. Some form unstable linkages with the proteinthat are subject to hydrolysis and therefore do not last very long inaqueous environments, such as in the blood stream. Some form more stablelinkages, but are subject to hydrolysis before the linkage is formed,which means that the reactive group on the PEG derivative may beinactivated before the protein can be attached. Some are somewhat toxicand are therefore less suitable for use in vivo. Some are too slow toreact to be practically useful. Some result in a loss of proteinactivity by attaching to sites responsible for the protein's activity.Some are not specific in the sites to which they will attach, which canalso result in a loss of desirable activity and in a lack ofreproducibility of results. In order to overcome the challengesassociated with modifying proteins with poly(ethylene glycol) moieties,PEG derivatives have been developed that are more stable (e.g., U.S.Pat. No. 6,602,498) or that react selectively with thiol moieties onmolecules and surfaces (e.g., U.S. Pat. No. 6,610,281). There is clearlya need in the art for PEG derivatives that are chemically inert inphysiological environments until called upon to react selectively toform stable chemical bonds.

Recently, an entirely new technology in the protein sciences has beenreported, which promises to overcome many of the limitations associatedwith site-specific modifications of proteins. Specifically, newcomponents have been added to the protein biosynthetic machinery of theprokaryote Escherichia coli (E. coli) (e.g., L. Wang, et al., (2001),Science 292:498-500) and the eukaryote Sacchromyces cerevisiae (S.cerevisiae) (e.g., J. Chin et al., Science 301:964-7 (2003)), which hasenabled the incorporation of non-genetically encoded amino acids toproteins in vivo. A number of new amino acids with novel chemical,physical or biological properties, including photoaffinity labels andphotoisomerizable amino acids, keto amino acids, and glycosylated aminoacids have been incorporated efficiently and with high fidelity intoproteins in E. coli and in yeast in response to the amber codon, TAG,using this methodology. See, e.g., J. W. Chin et al., (2002), Journal ofthe American Chemical Society 124:9026-9027; J. W. Chin, & P. G.Schultz, (2002), Chem Bio Chem 11:1135-1137; J. W. Chin, et al., (2002),PNAS United States of America 99:11020-11024: and, L. Wang, & P. G.Schultz, (2002), Chem. Comm., 1-10. These studies have demonstrated thatit is possible to selectively and routinely introduce chemicalfunctional groups, such as alkyne groups and azide moieties, that arenot found in proteins, that are chemically inert to all of thefunctional groups found in the 20 common, genetically-encoded aminoacids and that react efficiently and selectively to form stable covalentlinkages.

The ability to incorporate non-genetically encoded amino acids intoproteins permits the introduction of chemical functional groups thatcould provide valuable alternatives to the naturally-occurringfunctional groups, such as the epsilon —NH₂ of lysine, the sulfhydryl—SH of cysteine, the imino group of histidine, etc. Certain chemicalfunctional groups are known to be inert to the functional groups foundin the 20 common, genetically-encoded amino acids but react cleanly andefficiently to form stable linkages. Azide and acetylene groups, forexample, are known in the art to undergo a Huisgen [3+2] cycloadditionreaction in aqueous conditions in the presence of a catalytic amount ofcopper. See, e.g., Tornoe, et al., (2002) Org. Chem. 67:3057-3064; and,Rostovtsev, et al., (2002) Angew. Chem. Int. Ed. 41:2596-2599. Byintroducing an azide moiety into a protein structure, for example, oneis able to incorporate a functional group that is chemically inert toamines, sulfhydryls, carboxylic acids, hydroxyl groups found inproteins, but that also reacts smoothly and efficiently with anacetylene moiety to form a cycloaddition product. Importantly, in theabsence of the acetylene moiety, the azide remains chemically inert andunreactive in the presence of other protein side chains and underphysiological conditions.

While the art has disclosed compositions and methods for theintroduction of non-genetically encoded amino acids into proteinstructures, there has been no effort to develop PEG derivatives that arecapable of reacting efficiently and specifically with the new chemicalfunctionalities. Accordingly, there is a need in the art for new PEGderivatives that provide the benefits associated with PEG polymers(i.e., increased solubility, stability and half-life and diminishedimmunogenicity) but that provide greater selectively and versatility foruse in the modification of proteins with poly(ethylene glycol)derivatives. The present invention fulfils this and other needs.

SUMMARY OF THE INVENTION

The invention provides, in some embodiments, a water-soluble compoundthat includes a polymer and at least one terminal azide moiety. Thecompound or polymer may be selected from a wide range of suitable watersoluble compounds or polymers. In one embodiment the compounds orpolymers include but are not limited to nucleic acid molecules,polypeptides, charged compounds or polymers, and linear, branched ormulti-armed compounds or polymers. In another embodiment the polymerincludes but is not limited to poly(alkylene oxides), poly(oxyethylatedpolyols), and poly(olefinic alcohols). In some embodiments, the polymerincludes but is not limited to poly(ethylene glycol), poly(propyleneglycol), poly(oxyethylated glycerol), poly(oxyethylated sorbitol),poly(oxyethylated glucose), and poly(vinyl alcohol). The water-solublecompounds of the invention can be, for example, a polymer such aspoly(ethylene glycol)azide in which the azide moiety is covalentlyattached directly to a polymer backbone. Alternatively, the azide moietycan be covalently attached to the polymer backbone by a linker. In someembodiments, the polymer is a straight chain polymer and the compound isnot substituted with reactive functional groups beyond the azide moiety.In other embodiments, the polymer is a random or block copolymer orterpolymer.

In some embodiments, the invention provides water-soluble compounds thathave a dumbbell structure that includes: a) an azide moiety on at leasta first end of a polymer backbone; and b) at least a second functionalgroup on a second end of the polymer backbone. The second functionalgroup can be another azide moiety, or a different reactive group. Thesecond functional group, in some embodiments, is not reactive with azidemoieties. The invention provides, in some embodiments, water-solublecompounds that comprise at least one arm of a branched molecularstructure. For example, the branched molecular structure can bedendritic.

In some embodiments, the azide moiety forms a linkage with anotherreactive moiety which may be on a surface or in a molecule. For example,the reactive moiety can be an acetylene moiety. The azide moiety can belinked to said polymer by a linkage that includes a linker moiety. Inthese embodiments, the polymer can include at least a second functionalgroup other than the azide moiety for linking to said linker moiety. Forexample, the second functional group can be specific for nucleophilicdisplacement and the linker moiety can include a nucleophilic moietycapable of reacting with said functional group. In other embodiments,the second functional group is amine-specific and said linker moietyincludes an active amine moiety. As another example, the secondfunctional group can be specific for reaction at an electrophiliccarbonyl and said linker moiety can include an electrophilic carbonylmoiety. For example, the second functional group can be specific forreaction with an activated ester and the linker moiety can include anactivated ester. As another example, the second functional group can, bespecific for reaction with a ketone and said linker moiety includes anketone. In another embodiment, the second functional group is specificfor reaction with a thiol nucleophile and the linker moiety includes athiol nucleophile. The second functional group is, in some embodiments,specific for reaction with a hydroxyl nucleophile and the linker moietyincludes a hydroxyl nucleophile.

In some embodiments, the water-soluble compounds of the invention arestable in aqueous environments at a pH of about 11 or less.

Suitable linker moieties include, but are not limited to,—NH—CO—CH₂—CH₂—, —CO—NH—CH₂—CH₂—, —S—CH₂—CH₂—, and —O—CH₂—CH₂—.

The invention also provides water soluble activated polymers that arestable against hydrolysis, which activated polymers have the formula:

R—CH₂CH₂—(OCH₂CH₂)_(n)—N₃;

wherein

n is from about 5 to 3,000, and R is a capping group, a functionalgroup, or a leaving group that can be the same or different from theazide. R can be, for example, a functional group selected from the groupconsisting of hydroxyl, protected hydroxyl, alkoxyl,N-hydroxysuccinimidyl ester, 1-benzotriazolyl ester,N-hydroxysuccinimidyl carbonate, 1-benzotriazolyl carbonate, acetal,aldehyde, aldehyde hydrates, alkenyl, acrylate, methacrylate,acrylamide, active sulfone, amine, aminooxy, protected amine, hydrazide,protected hydrazide, protected thiol, carboxylic acid, protectedcarboxylic acid, isocyanate, isothiocyanate, maleimide, vinylsulfone,dithiopyridine, vinylpyridine, iodoacetamide, epoxide, glyoxals, diones,mesylates, tosylates, and tresylate, alkene, and ketone.

The invention also provides a water-soluble compound that includes apolymer and at least one acetylene moiety, wherein the polymer isselected from the group consisting of poly(alkylene oxides),poly(oxyethylated polyols), and poly(olefinic alcohols). The polymer, insome embodiments, is selected from the group consisting of poly(ethyleneglycol), poly(propylene glycol), poly(oxyethylated glycerol),poly(oxyethylated sorbitol), poly(oxyethylated glucose), and poly(vinylalcohol). For example, the compound can be a poly(ethyleneglycol)acetylene, wherein the acetylene moiety is covalently bound to apolymer backbone via an ether linkage. In some embodiments, the compoundis poly(ethylene glycol)acetylene and the acetylene moiety is covalentlyattached directly to a polymer backbone. Alternatively, the acetylenemoiety can be covalently attached to the polymer backbone by a linker.In some embodiments, the polymer is a straight chain polymer and thecompound is not substituted beyond the acetylene moiety. In otherembodiments, the polymer is a random or block copolymer or terpolymer.

In some embodiments, the invention provides water-soluble compounds thathave a dumbbell structure that includes: a) an acetylene moiety on atleast a first end of a polymer backbone; and b) at least a secondfunctional group on a second end of the polymer backbone. The secondfunctional group can be another acetylene moiety, or a differentreactive group. The second functional group, in some embodiments, is notreactive with acetylene moieties. The invention provides, in someembodiments, water-soluble compounds that comprise at least one arm of abranched molecular structure. For example, the branched molecularstructure can be dendritic.

In some embodiments, the acetylene moiety forms a linkage with areactive moiety on a surface or in a molecule. For example, the reactivemoiety can be an acetylene moiety. The acetylene moiety can be linked tosaid polymer by a linkage that includes a linker moiety. In theseembodiments, the polymer can include at least a second functional groupother than the acetylene moiety for linking to said linker moiety. Forexample, the second functional group can be specific for nucleophilicdisplacement and the linker moiety can include a nucleophilic moietycapable of reacting with said functional group. In other embodiments,the second functional group is amine-specific and said linker moietyincludes an active amine moiety. As another example, the secondfunctional group can be specific for reaction at an electrophiliccarbonyl and said linker moiety can include an electrophilic carbonylmoiety. For example, the second functional group can be specific forreaction with an activated ester and the linker moiety can include anactivated ester. As another example, the second functional group can bespecific for reaction with a ketone and said linker moiety includes anketone. In another embodiment, the second functional group is specificfor reaction with a thiol nucleophile and the linker moiety includes athiol nucleophile. The second functional group is, in some embodiments,specific for reaction with a hydroxyl nucleophile and the linker moietyincludes a hydroxyl nucleophile.

DETAILED DESCRIPTION

This invention provides a highly efficient method for the selectivemodification of proteins with derivatives of water soluble compounds orpolymers, including but not limited to PEG derivatives. The derivativesof water soluble compounds or polymers of the present invention may beadded to any protein comprising an appropriate functional group. Theappropriate functional group may be added to a polypeptide by modifyingone or more amino acids of the molecule at a suitable location includingbut not limited to the amino-terminus, carboxyl-terminus, or within thepolypeptide chain. The appropriate functional group may be added to apolypeptide by modifying one or more side chains of one or morenaturally occurring amino acids. Alternatively, the appropriatefunctional groups may be incorporated into a polypeptide using standardchemical polypeptide synthesis with amino acids having the functionalgroup present, or by the selective incorporation of non-geneticallyencoded amino acids, e.g., those amino acids containing an azide oracetylene moiety, into proteins in response to a selector codon. Onceincorporated, the amino acid side chains can then be modified by anysuitably reactive water soluble compound or polymer. The suitablyreactive compounds or polymers include but are not limited to PEGderivatives. The suitable reactions that may be utilized to attach thewater soluble compounds or derivatives to the appropriate functionalgroups of the polypeptide include but are not limited to, e.g., aHuisgen [3+2] cycloaddition reaction (see, e.g., Padwa, A. inComprehensive Organic Synthesis, Vol. 4, (1991) Ed. Trost, B. M.,Pergamon, Oxford, p. 1069-1109; and, Huisgen, R. in 1,3-DipolarCycloaddition Chemistry, (1984) Ed. Padwa, A., Wiley, New York, p.1-176) with, e.g., acetylene or azide derivatives, respectively.

Because the method involves a cycloaddition rather than a nucleophilicsubstitution reaction, proteins can be modified with extremely highselectivity. The reaction can be carried out at room temperature inaqueous conditions with excellent regioselectivity (1,4>1,5) by theaddition of catalytic amounts of Cu(I) salts to the reaction mixture.See, e.g., Tornoe, et al., (2002) Org. Chem. 67:3057-3064; and,Rostovtsev, et al., (2002) Angew. Chem. Int. Ed. 41:2596-2599. Amolecule that can be added to a protein of the invention through a [3+2]cycloaddition includes virtually any molecule with an azido or acetylenederivative. These molecules may be added to the appropriate functionalgroup on a modified naturally occurring amino acid or an unnatural aminoacid with an acetylene group, e.g., p-propargyloxyphenylalanine, orazido group, e.g., p-azido-phenylalanine, respectively.

The resulting five-membered ring that results from the Huisgen [3+2]cycloaddition is not generally reversible in reducing environments andis stable against hydrolysis for extended periods in aqueousenvironments. Consequently, the physical and chemical characteristics ofa wide variety of substances can be modified under demanding aqueousconditions with the active PEG derivatives of the present invention.Even more important, because the azide and acetylene moieties arespecific for one another (and do not, for example, react with the sidechain functional groups of any of the 20 common, genetically-encodedamino acids), proteins can be modified in one or more specific siteswith extremely high selectivity.

The invention also provides water soluble and hydrolytically stablederivatives of compounds and polymers, including but not limited to PEGderivatives and related hydrophilic polymers having one or moreacetylene or azide moieties. The PEG polymer derivatives that containacetylene moieties are highly selective for coupling with azide moietiesthat have been introduced selectively into proteins in response to aselector codon. Similarly, PEG polymer derivatives that contain azidemoieties are highly selective for coupling with acetylene moieties thathave been introduced selectively into proteins in response to a selectorcodon.

More specifically, the azide moieties comprise alkyl azides, aryl azidesand derivatives of these azides. The derivatives of the alkyl and arylazides can include other substituents so long as the acetylene-specificreactivity is maintained. The acetylene moieties comprise alkyl and arylacetylenes and derivatives of each. The derivatives of the alkyl andaryl acetylenes can include other substituents so long as theazide-specific reactivity is maintained.

The invention includes conjugates of substances having azide oracetylene moieties with water soluble compounds or polymers, such as butnot limited to, PEG polymer derivatives having the correspondingacetylene or azide moieties. For example, a PEG polymer containing anazide moiety can be coupled to a biologically active molecule at aposition in the protein that contains a non-genetically encoded aminoacid bearing an acetylene functionality. The linkage by which the PEGand the biologically active molecule are coupled includes the Huisgen[3+2] cycloaddition product.

It is well established in the art that PEG can be used to modify thesurfaces of biomaterials (see, e.g., U.S. Pat. No. 6,610,281). Theinvention also includes biomaterials comprising a surface having one ormore reactive azide or acetylene sites and one or more of the azide- oracetylene-containing polymers of the invention coupled to the surfacevia the Huisgen [3+2] cycloaddition linkage. Biomaterials and othersubstances can also be coupled to the azide- or acetylene-activatedpolymer derivatives through a linkage other than the azide or acetylenelinkage, such as through a linkage comprising a carboxylic acid, amine,alcohol or thiol moiety, to leave the azide or acetylene moietyavailable for subsequent reactions.

The invention includes a method of synthesizing the azide- and acetylenecontaining compounds or polymers of the invention. In the case of theazide-containing compound or polymer derivative, the azide can be bondeddirectly to a carbon atom of the polymer. Alternatively, theazide-containing compound or polymer derivative can be prepared byattaching a linking agent that has the azide moiety at one terminus to aconventional activated polymer so that the resulting polymer has theazide moiety at its terminus or any other desired location in themolecule. In the case of the acetylene-containing compound or polymerderivative, the acetylene can be bonded directly to a carbon atom of thepolymer. Alternatively, the acetylene-containing compound or polymerderivative can be prepared by attaching a linking agent that has theacetylene moiety at one terminus to a conventional activated polymer sothat the resulting polymer has the acetylene moiety within the moleculeor at its terminus.

More specifically, in the case of the azide-containing water solublecompound or polymer such as a PEG derivative, a water soluble polymerhaving at least one active hydroxyl moiety undergoes a reaction toproduce a substituted polymer having a more reactive moiety, such as amesylate, tresylate, tosylate or halogen leaving group, thereon. Thepreparation and use of PEG or other compound or polymer derivativescontaining sulfonyl acid halides, halogen atoms and other leaving groupsare well known to the skilled artisan. The resulting substituted polymerthen undergoes a reaction to substitute for the more reactive moiety anazide moiety at the terminus of the polymer. Alternatively, a watersoluble polymer having at least one active nucleophilic or electrophilicmoiety undergoes a reaction with a linking agent that has an azide atone terminus so that a covalent bond is formed between the polymer andthe linking agent and the azide moiety is positioned at the terminus ofthe polymer. Nucleophilic and electrophilic moieties, including amines,thiols, hydrazides, hydrazines, alcohols, carboxylates, aldehydes,ketones, thioesters and the like, are well known to the skilled artisan.

More specifically, in the case of the acetylene-containing compound orpolymer derivative, a water soluble polymer having at least one activehydroxyl moiety undergoes a reaction to displace a halogen or otheractivated leaving group from a precursor that contains an acetylenemoiety. Alternatively, a water soluble polymer having at least oneactive nucleophilic or electrophilic moiety undergoes a reaction with alinking agent that has an acetylene at one terminus so that a covalentbond is formed between the polymer and the linking agent and theacetylene moiety is positioned at the terminus of the polymer. The useof halogen moieties, activated leaving group, nucleophilic andelectrophilic moieties in the context of organic synthesis and thepreparation and use of PEG derivatives is well established topractitioners in the art.

Thus the invention provides a method for the selective modification ofproteins with water soluble compounds or polymers, such as PEG,derivatives containing an azide or acetylene moiety. The azide- andacetylene-containing PEG derivatives can be used to modify theproperties of surfaces and molecules where biocompatibility, stability,solubility and lack of immunogenicity are important, while at the sametime providing a more selective means of attaching the PEG derivativesto proteins than has been known in the art.

DEFINITIONS

The terms “functional group”, “active moiety”, “activating group”,“leaving group”, “reactive site”, “chemically reactive group” and“chemically reactive moiety” are used in the art and herein to refer todistinct, definable portions or units of a molecule. The terms are usedherein to indicate the portions of molecules that perform some functionor activity and are reactive with other molecules.

The term “linkage” or “linker” is used herein to refer to groups orbonds that normally are formed as the result of a chemical reaction andtypically are covalent linkages. Hydrolytically stable linkages meansthat the linkages are substantially stable in water and do not reactwith water at useful pHs, e.g., under physiological conditions for anextended period of time, perhaps even indefinitely. Hydrolyticallyunstable or degradable linkages means that the linkages are degradablein water or in aqueous solutions, including for example, blood.Enzymatically unstable or degradable linkages means that the linkage canbe degraded by one or more enzymes. As understood in the art, PEG andrelated polymers may include degradable linkages in the polymer backboneor in the linker group between the polymer backbone and one or more ofthe terminal functional groups of the polymer molecule. For example,ester linkages formed by the reaction of PEG carboxylic acids oractivated PEG carboxylic acids with alcohol groups on a biologicallyactive agent generally hydrolyze under physiological conditions torelease the agent. Other hydrolytically degradable linkages includecarbonate linkages; imine linkages resulted from reaction of an amineand an aldehyde; phosphate ester linkages formed by reacting an alcoholwith a phosphate group; hydrazone linkages which are reaction product ofa hydrazide and an aldehyde; acetal linkages that are the reactionproduct of an aldehyde and an alcohol; orthoester linkages that are thereaction product of a formate and an alcohol; peptide linkages formed byan amine group, e.g., at an end of a polymer such as PEG, and a carboxylgroup of a peptide; and oligonucleotide linkages formed by aphosphoramidite group, e.g., at the end of a polymer, and a 5′ hydroxylgroup of an oligonucleotide.

The term “biologically active molecule”, “biologically active moiety” or“biologically active agent” when used herein means any substance whichcan affect any physical or biochemical properties of a biologicalorganism, including but not limited to viruses, bacteria, fungi, plants,animals, and humans. In particular, as used herein, biologically activemolecules include any substance intended for diagnosis, cure mitigation,treatment, or prevention of disease in humans or other animals, or tootherwise enhance physical or mental well-being of humans or animals.Examples of biologically active molecules include, but are not limitedto, peptides, proteins, enzymes, small molecule drugs, dyes, lipids,nucleosides, oligonucleotides, cells, viruses, liposomes, microparticlesand micelles. Classes of biologically active agents that are suitablefor use with the invention include, but are not limited to, antibiotics,fungicides, anti-viral agents, anti-inflammatory agents, anti-tumoragents, cardiovascular agents, anti-anxiety agents, hormones, growthfactors, steroidal agents, and the like.

The terms “alkyl,” “alkene,” and “alkoxy” include straight chain andbranched alkyl, alkene, and alkoxy, respectively. The term “lower alkyl”refers to C₁-C₆ alkyl. The term “alkoxy” refers to oxygen substitutedalkyl, for example, of the formulas —OR or —ROR₁, wherein R and R₁ areeach independently selected alkyl. The terms “substituted alkyl” and“substituted alkene” refer to alkyl and alkene, respectively,substituted with one or more non-interfering substituents, such as butnot limited to, C₃-C₆ cycloalkyl, e.g., cyclopropyl, cyclobutyl, and thelike; acetylene; cyano; alkoxy, e.g., methoxy, ethoxy, and the like;lower alkanoyloxy, e.g., acetoxy; hydroxy; carboxyl; amino; loweralkylamino, e.g., methylamino; ketone; halo, e.g. chloro or bromo;phenyl; substituted phenyl, and the like. The term “halogen” includesfluorine, chlorine, iodine and bromine.

“Aryl” means one or more aromatic rings, each of 5 or 6 carbon atoms.Multiple aryl rings may be fused, as in naphthyl or unfused, as inbiphenyl. Aryl rings may also be fused or unfused with one or morecyclic hydrocarbon, heteroaryl, or heterocyclic rings.

“Substituted aryl” is aryl having one or more non-interfering groups assubstituents.

The term “substituents” includes but is not limited to non-interferingsubstituents. “Non-interfering substituents” are those groups that yieldstable compounds. Suitable non-interfering substituents or radicalsinclude, but are not limited to, halo, C.sub.1-C.sub.10 alkyl, C₂-C₁₀alkenyl, C₂-C₁₀ alkynyl, C₁-C₁₀ alkoxy, C₁-C₁₂ aralkyl, C₁-C₁₂ alkaryl,C₃-C₁₂ cycloalkyl, C₃-C₁₂ cycloalkenyl, phenyl, substituted phenyl,toluoyl, xylenyl, biphenyl, C₂-C₁₂ alkoxyalkyl, C₂-C₁₂ alkoxyaryl,C₇-C₁₂ aryloxyalkyl, C₇-C₁₂ oxyaryl, C₁-C₆ alkylsulfinyl, C₁-C₁₀alkylsulfonyl, —(CH₂)_(m)—O—(C₁-C₁₀ alkyl) wherein m is from 1 to 8,aryl, substituted aryl, substituted alkoxy, fluoroalkyl, heterocyclicradical, substituted heterocyclic radical, nitroalkyl, —NO₂, —CN,—NRC(O)—(C₁-C₁₀ alkyl), —C(O)—(C₁-C₁₀ alkyl), C₂-C₁₀ alkyl thioalkyl,—C(O)O—(C₁-C₁₀ alkyl), —OH, —SO₂, .dbd.S, —COOH, —NR₂, carbonyl,—C(O)—(C₁-C₁₀ alkyl)-CF3, —C(O)—CF3, —C(O)NR2, —(C₁-C₁₀ aryl)—S—(C₆-C₁₀aryl), —C(O)—(C₁-C₁₀ aryl), —(CH₂)_(m)—O—(—(CH₂)_(m)—O—(C₁-C₁₀ alkyl)wherein each m is from 1 to 8, —C(O)NR.sub.2, —C(S)NR₂, SO₂NR₂,—NRC(O)NR₂, —NRC(S)NR₂, salts thereof, and the like. Each R as usedherein is H, alkyl or substituted alkyl, aryl or substituted aryl,aralkyl, or alkaryl.

The invention provides azide- and acetylene-containing polymerderivatives comprising a water soluble polymer backbone having anaverage molecular weight from about 800 Da to about 100,000 Da. Thepolymer backbone of the water-soluble polymer can be any suitablecompound or polymer, including but not limited to nucleic acidmolecules, polypeptides, charged compounds or polymers, linear, branchedor multi-armed compounds or polymers, and poly(ethylene glycol) (i.e.,PEG). However, it should be understood that other compounds andpolymers, including but not limited to PEG-related molecules such aspoly(dextran) and poly(propylene glycol), are also suitable for use inthe practice of this invention and that the use of the term PEG orpoly(ethylene glycol) is intended to be inclusive and not exclusive inthis respect. The term PEG includes poly(ethylene glycol) in any of itsforms, including bifunctional PEG, multiarmed PEG, forked PEG, branchedPEG, pendent PEG (i.e. PEG or related polymers having one or morefunctional groups pendent to the polymer backbone), or PEG withdegradable linkages therein.

As used herein, the term “water soluble polymer” refers to any polymerthat is soluble in aqueous solvents. Linkage of water soluble polymersto a therapeutic polypeptide can result in changes including, but notlimited to, increased or modulated serum half-life, or increased ormodulated therapeutic half-life relative to the unmodified form,modulated immunogenicity, modulated physical association characteristicssuch as aggregation and multimer formation, altered receptor binding andaltered receptor dimerization or multimerization. The water solublepolymer may or may not have its own biological activity. Suitablepolymers include, but are not limited to, polyethylene glycol,polyethylene glycol propionaldehyde, mono C1-C10 alkoxy or aryloxyderivatives thereof (described in U.S. Pat. No. 5,252,714 which isincorporated by reference herein), monomethoxy-polyethylene glycol,polyvinyl pyrrolidone, polyvinyl alcohol, polyamino acids, divinylethermaleic anhydride, N-(2-Hydroxypropyl)-methacrylamide, dextran, dextranderivatives including dextran sulfate, polypropylene glycol,polypropylene oxide/ethylene oxide copolymer, polyoxyethylated polyol,heparin, heparin fragments, polysaccharides, oligosaccharides, glycans,cellulose and cellulose derivatives, including but not limited tomethylcellulose and carboxymethyl cellulose, starch and starchderivatives, polypeptides, polyalkylene glycol and derivatives thereof,copolymers of polyalkylene glycols and derivatives thereof, polyvinylethyl ethers, and alpha-beta-poly[(2-hydroxyethyl)-DL-aspartamide, andthe like, or mixtures thereof. Examples of such water soluble polymersinclude but are not limited to polyethylene glycol and serum albumin.

As used herein, the term “polyalkylene glycol” refers to polyethyleneglycol, polypropylene glycol, polybutylene glycol, and derivativesthereof. The term “polyalkylene glycol” encompasses both linear andbranched polymers and average molecular weights of between 1 kDa and 100kDa. Other exemplary embodiments are listed, for example, in commercialsupplier catalogs, such as Shearwater Corporation's catalog“Polyethylene Glycol and Derivatives for Biomedical Applications”(2001).

PEG is typically clear, colorless, odorless, soluble in water, stable toheat, inert to many chemical agents, does not hydrolyze or deteriorate,and is generally non-toxic. Poly(ethylene glycol) is considered to bebiocompatible, which is to say that PEG is capable of coexistence withliving tissues or organisms without causing harm. More specifically, PEGis substantially non-immunogenic, which is to say that PEG does not tendto produce an immune response in the body. When attached to a moleculehaving some desirable function in the body, such as a biologicallyactive agent, the PEG tends to mask the agent and can reduce oreliminate any immune response so that an organism can tolerate thepresence of the agent. PEG conjugates tend not to produce a substantialimmune response or cause clotting or other undesirable effects. PEGhaving the formula —CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—, where n is from about3 to about 4000, typically from about 20 to about 2000, is one usefulpolymer in the practice of the invention. PEG having a molecular weightof from about 800 Da to about 100,000 Da are particularly useful as thepolymer backbone.

The polymer backbone can be linear or branched. Branched polymerbackbones are generally known in the art. Typically, a branched polymerhas a central branch core moiety and a plurality of linear polymerchains linked to the central branch core. PEG is commonly used inbranched forms that can be prepared by addition of ethylene oxide tovarious polyols, such as glycerol, glycerol oligomers, pentaerythritoland sorbitol. The central branch moiety can also be derived from severalamino acids, such as lysine. The branched poly(ethylene glycol) can berepresented in general form as R(-PEG-OH)_(m) in which R is derived froma core moiety, such as glycerol, glycerol oligomers, or pentaerythritol,and m represents the number of arms. Multi-armed PEG molecules, such asthose described in U.S. Pat. Nos. 5,932,462 5,643,575; 5,229,490;4,289,872; U.S. Pat. Appl. 2003/0143596; WO 96/21469; and WO 93/21259,each of which is incorporated by reference herein in its entirety, canalso be used as the polymer backbone.

Branched PEG can also be in the form of a forked PEG represented byPEG(-YCHZ₂)_(n), where Y is a linking group and Z is an activatedterminal group linked to CH by a chain of atoms of defined length.

Yet another branched form, the pendant PEG, has reactive groups, such ascarboxyl, along the PEG backbone rather than at the end of PEG chains.

In addition to these forms of PEG, the polymer can also be prepared withweak or degradable linkages in the backbone. For example, PEG can beprepared with ester linkages in the polymer backbone that are subject tohydrolysis. As shown below, this hydrolysis results in cleavage of thepolymer into fragments of lower molecular weight:

-PEG-CO₂-PEG-+H₂O→PEG-CO₂H+HO-PEG-

It is understood by those skilled in the art that the term poly(ethyleneglycol) or PEG represents or includes all the above forms.

Many other polymers are also suitable for the invention. Polymerbackbones that are water-soluble, with from 2 to about 300 termini, areparticularly useful in the invention. Examples of suitable polymersinclude, but are not limited to, other poly(alkylene glycols), such aspoly(propylene glycol) (“PPG”), copolymers thereof (e.g. copolymers ofethylene glycol and propylene glycol), terpolymers thereof, mixturesthereof, and the like. Although the molecular weight of each chain ofthe polymer backbone can vary, it is typically in the range of fromabout 800 Da to about 100,000 Da, often from about 6,000 Da to about80,000 Da.

Those of ordinary skill in the art will recognize that the foregoinglist for substantially water soluble backbones is by no means exhaustiveand is merely illustrative, and that all polymeric materials having thequalities described above are contemplated.

The polymer derivatives of the invention are “multi-functional”, meaningthat the polymer backbone has at least two termini, and possibly as manyas about 300 termini, functionalized or activated with a functionalgroup. Multifunctional polymer derivatives include linear polymershaving two termini, each terminus being bonded to a functional groupwhich may be the same or different.

In one embodiment, the polymer derivative has the structure:

X-A-POLY-B-N═N═N

wherein:

-   -   N═N═N is an azide moiety;    -   B is a linking moiety, which may be present or absent;    -   POLY is a water-soluble non-antigenic polymer;    -   A is a linking moiety, which may be present or absent and which        may be the same as B or different; and    -   X is a second functional group.

Examples of a linking moiety for A and B include amultiply-functionalized alkyl group containing up to 18, and morepreferably between 1-10 carbon atoms. A heteroatom such as nitrogen,oxygen or sulfur may be included with the alkyl chain. The alkyl chainmay also be branched at a heteroatom. Other examples of a linking moietyfor A and B include a multiply functionalized aryl group, containing upto 10 and more preferably 5-6 carbon atoms. The aryl group may besubstituted with one more carbon atoms, nitrogen, oxygen or sulfuratoms. Other examples of suitable linking groups include those linkinggroups described in U.S. Pat. Nos. 5,932,462 and 5,643,575 and U.S. Pat.Appl. 2003/0143596, each of which is incorporated by reference herein inits entirety. Those of ordinary skill in the art will recognize that theforegoing list for linking moieties is by no means exhaustive and merelyillustrative, and that all linking moieties having the qualitiesdescribed above are contemplated.

Examples of suitable functional groups for use as X include hydroxyl,protected hydroxyl, alkoxyl, active ester, such as N-hydroxysuccinimidylesters and 1-benzotriazolyl esters, active carbonate, such asN-hydroxysuccinimidyl carbonates and 1-benzotriazolyl carbonates,acetal, aldehyde, aldehyde hydrates, alkenyl, acrylate, methacrylate,acrylamide, active sulfone, amine, aminooxy, protected amine, hydrazide,protected hydrazide, protected thiol, carboxylic acid, protectedcarboxylic acid, isocyanate, isothiocyanate, maleimide, vinylsulfone,dithiopyridine, vinylpyridine, iodoacetamide, epoxide, glyoxals, diones,mesylates, tosylates, and tresylate, alkene, ketone, and azide. As wouldbe understood, the selected X moiety should be compatible with the azidegroup so that reaction with the azide group does not occur. Theazide-containing polymer derivatives may be homobifunctional, meaningthat the second functional group (i.e., X) is also an azide moiety, orheterobifunctional, meaning that the second functional group is adifferent functional group.

As would be understood in the art, the term “protected” refers to thepresence of a protecting group or moiety that prevents reaction of thechemically reactive functional group under certain reaction conditions.The protecting group will vary depending on the type of chemicallyreactive group being protected. For example, if the chemically reactivegroup is an amine or a hydrazide, the protecting group can be selectedfrom the group of tert-butyloxycarbonyl (t-Boc) and9-fluorenylmethoxycarbonyl (Fmoc). If the chemically reactive group is athiol, the protecting group can be orthopyridyldisulfide. If thechemically reactive group is a carboxylic acid, such as butanoic orpropionic acid, or a hydroxyl group, the protecting group can be benzylor an alkyl group such as methyl, ethyl, or tert-butyl. Other protectinggroups known in the art may also be used in the invention.

Specific examples of terminal functional groups in the literatureinclude N-succinimidyl carbonate (see e.g., U.S. Pat. Nos. 5,281,698,5,468,478), amine (see, e.g., Buckmann et al. Makromol. Chem. 182:1379(1981), Zaplipsky et al. Eur. Polym. J. 19:1177 (1983)), hydrazide (See,e.g., Andresz et al. Makromol. Chem. 179:301 (1978)), succinimidylpropionate and succinimidyl butanoate (see, e.g., Olson et al. inPolyethylene glycol) Chemistry & Biological Applications, pp 170-181,Harris & Zaplipsky Eds., ACS, Washington, D.C., 1997; see also U.S. Pat.No. 5,672,662), succinimidyl succinate (See, e.g., Abuchowski et al.Cancer Biochem. Biophys. 7:175 (1984) and Joppich et al. Macrolol. Chem.180:1381 (1979), succinimidyl ester (see, e.g., U.S. Pat. No.4,670,417), benzotriazole carbonate (see, e.g., U.S. Pat. No.5,650,234), glycidyl ether (see, e.g., Pitha et al. Eur. J. Biochem.94:11 (1979), Elling et al., Biotech. Appl. Biochem. 13:354 (1991),oxycarbonylimidazole (see, e.g., Beauchamp, et al., Anal. Biochem.131:25 (1983), Tondelli et al. J. Controlled Release 1:251 (1985)),p-nitrophenyl carbonate (see, e.g., Veronese, et al., Appl. Biochem.Biotech., 11: 141 (1985); and Sartore et al., Appl. Biochem. Biotech.,27:45 (1991)), aldehyde (see, e.g., Harris et al. J. Polym. Sci. Chem.Ed. 22:341 (1984), U.S. Pat. No. 5,824,784, U.S. Pat. No. 5,252,714),maleimide (see, e.g., Goodson et al. Bio/Technology 8:343 (1990), Romaniet al. in Chemistry of Peptides and Proteins 2:29 (1984)), and Kogan,Synthetic Comm. 22:2417 (1992)), orthopyridyl-disulfide (see, e.g.,Woghiren, et al. Bioconj. Chem. 4:314 (1993)), acrylol (see, e.g.,Sawhney et al., Macromolecules, 26:581 (1993)), vinylsulfone (see, e.g.,U.S. Pat. No. 5,900,461). All of the above references are incorporatedherein by reference.

In a preferred embodiment, the polymer derivatives of the inventioncomprise a polymer backbone having the structure:

X—CH₂CH₂O—(CH₂CH₂O)n—CH₂CH₂—N═N═N

wherein:

X is a functional group as described above; and

n is about 20 to about 4000.

In another embodiment, the polymer derivatives of the invention comprisea polymer backbone having the structure:

X—CH₂CH₂O—(CH₂CH₂O)n—CH₂CH₂—O—(CH₂)_(m)-W-N═N═N

wherein:

-   -   W is an aliphatic or aromatic linker moiety comprising between        1-10 carbon atoms;    -   n is about 20 to about 4000; and    -   X is a functional group as described above.

The azide-containing PEG derivatives of the invention can be prepared byat least two methods. In one method, shown below, a water solublepolymer backbone having an average molecular weight from about 800 Da toabout 100,000 Da, the polymer backbone having a first terminus bonded toa first functional group and a second terminus bonded to a suitableleaving group, is reacted with an azide anion (which may be paired withany of a number of suitable counter-ions, including sodium, potassium,tert-butylammonium and so forth). The leaving group undergoes anucleophilic displacement and is replaced by the azide moiety, affordingthe desired azide-containing PEG polymer:

X-PEG-L+N₃ ⁻→X-PEG-N₃

As shown, a suitable polymer backbone for use in the reaction has theformula X-PEG-L, wherein PEG is poly(ethylene glycol) and X is afunctional group which does not react with azide groups and L is asuitable leaving group. Examples of suitable functional groups includehydroxyl, protected hydroxyl, acetal, alkenyl, amine, aminooxy,protected amine, protected hydrazide, protected thiol, carboxylic acid,protected carboxylic acid, maleimide, dithiopyridine, and vinylpyridine,and ketone. Examples of suitable leaving groups include chloride,bromide, iodide, mesylate, tresylate, and tosylate.

In a second method for preparation of the azide-containing polymerderivatives of the invention, a linking agent bearing an azidefunctionality is contacted with a water soluble polymer backbone havingan average molecular weight from about 800 Da to about 100,000 Da,wherein the linking agent bears a chemical functionality that will reactselectively with a chemical functionality on the PEG polymer, to form anazide-containing polymer derivative product wherein the azide isseparated from the polymer backbone by a linking group.

An exemplary reaction scheme is shown below:

X-PEG-M+N-linker-N═N═N→PG-X-PEG-linker-N═N═N

wherein: PEG is poly(ethylene glycol) and X is a capping group such asalkoxy or a functional group as described above; and M is a functionalgroup that is not reactive with the azide functionality but that willreact efficiently and selectively with the N functional group.

Examples of suitable functional groups include: M being a carboxylicacid, carbonate or active ester if N is an amine; M being a ketone if Nis a hydrazide or aminooxy moiety; M being a, leaving group if N is anucleophile.

Purification of the crude product can usually be accomplished byprecipitation of the product followed by chromatography, if necessary.

A more specific example is shown below in the case of PEG diamine, inwhich one of the amines is protected by a protecting group moiety suchas tert-butyl-Boc and the resulting mono-protected PEG diamine isreacted with a linking moiety that bears the azide functionality:BocHN-PEG-NH₂+HO₂C—(CH₂)₃—N═N═N.

In this instance, the amine group can be coupled to the carboxylic acidgroup using a variety of activating agents such as thionyl chloride orcarbodiimide reagents and N-hydroxysuccinimide or N-hydroxybenzotriazoleto create an amide bond between the monoamine PEG derivative and theazide-bearing linker moiety. After successful formation of the amidebond, the resulting N-tert-butyl-Boc-protected azide-containingderivative can be used directly to modify bioactive molecules or it canbe further elaborated to install other useful functional groups. Forinstance, the N-t-Boc group can be hydrolyzed by treatment with strongacid to generate an omega-amino-PEG-azide. The resulting amine can beused as a synthetic handle to install other useful functionality such asmaleimide groups, activated disulfides, activated esters and so forthfor the creation of valuable heterobifunctional reagents.

Heterobifunctional derivatives are particularly useful when it isdesired to attach different molecules to each terminus of the polymer.For example, the omega.-N-amino-N-azido PEG would allow the attachmentof a molecule having an activated electrophilic group, such as analdehyde, ketone, activated ester, activated carbonate and so forth, toone terminus of the PEG and a molecule having an acetylene group to theother terminus of the PEG.

In another embodiment of the invention, the polymer derivative has thestructure:

X-A-POLY-B-C≡C—R

wherein:

-   -   R can be either H or an alkyl, alkene, alkyoxy, or aryl or        substituted aryl group;    -   B is a linking moiety, which may be present or absent;    -   POLY is a water-soluble non-antigenic polymer;    -   A is a linking moiety, which may be present or absent and which        may be the same as B or different; and    -   X is a second functional group.

Examples of a linking moiety for A and B include amultiply-functionalized alkyl group containing up to 18, and morepreferably between 1-10 carbon atoms. A heteroatom such as nitrogen,oxygen or sulfur may be included with the alkyl chain. The alkyl chainmay also be branched at a heteroatom. Other examples of a linking moietyfor A and B include a multiply functionalized aryl group, containing upto 10 and more preferably 5-6 carbon atoms. The aryl group may besubstituted with one more carbon atoms, nitrogen, oxygen or sulfuratoms. Other examples of suitable linking groups include those linkinggroups described in U.S. Pat. Nos. 5,932,462 and 5,643,575 and U.S. Pat.Appl. 2003/0143596, each of which is incorporated by reference herein inits entirety. Those of ordinary skill in the art will recognize that theforegoing list for linking moieties is by no means exhaustive and merelyillustrative, and that all linking moieties having the qualitiesdescribed above are contemplated.

Examples of suitable functional groups for use as X include hydroxyl,protected hydroxyl, alkoxyl, active ester, such as N-hydroxysuccinimidylesters and 1-benzotriazolyl esters, active carbonate, such asN-hydroxysuccinimidyl carbonates and 1-benzotriazolyl carbonates,acetal, aldehyde, aldehyde hydrates, alkenyl, acrylate, methacrylate,acrylamide, active sulfone, amine, aminooxy, protected amine, hydrazide,protected hydrazide, protected thiol, carboxylic acid, protectedcarboxylic acid, isocyanate, isothiocyanate, maleimide, vinylsulfone,dithiopyridine, vinylpyridine, iodoacetamide, epoxide, glyoxals, diones,mesylates, tosylates, and tresylate, alkene, ketone, and acetylene. Aswould be understood, the selected X moiety should be compatible with theacetylene group so that reaction with the acetylene group does notoccur. The acetylene-containing polymer derivatives may behomobifunctional, meaning that the second functional group (i.e., X) isalso an acetylene moiety, or heterobifunctional, meaning that the secondfunctional group is a different functional group.

In another preferred embodiment, the polymer derivatives of theinvention comprise a polymer backbone having the structure:

X—CH₂CH₂O—(CH₂CH₂O)n—CH₂CH₂—O—(CH₂)_(m)—C≡CH

wherein:

X is a functional group as described above;

n is about 20 to about 4000; and

m is between 1 and 10.

Specific examples of each of the heterobifunctional PEG polymers areshown below.

The acetylene-containing PEG derivatives of the invention can beprepared by at least two methods. In a first method, a water solublepolymer backbone having an average molecular weight from about 800 Da toabout 100,000 Da, the polymer backbone having a first terminus bonded toa first functional group and a second terminus bonded to a suitablenucleophilic group, is reacted with a compound that bears both anacetylene functionality and a leaving group that is suitable forreaction with the nucleophilic group on the PEG. When the PEG polymerbearing the nucleophilic moiety and the molecule bearing the leavinggroup are combined, the leaving group undergoes a nucleophilicdisplacement and is replaced by the nucleophilic moiety, affording thedesired acetylene-containing polymer.

X-PEG-Nu+L-A-C→X-PEG-Nu-A-C≡CR′

As shown, a preferred polymer backbone for use in the reaction has theformula X-PEG-Nu, wherein PEG is poly(ethylene glycol), Nu is anucleophilic moiety and X is a functional group that does not react withNu, L or the acetylene functionality.

Examples of Nu include amine, alkoxy, aryloxy, sulfhydryl, imino,carboxylate, hydrazide, aminoxy groups that would react primarily via aSN2-type mechanism. Additional examples of Nu groups include thosefunctional groups that would react primarily via an nucleophilicaddition reaction. Examples of L groups include chloride, bromide,iodide, mesylate, tresylate, and tosylate and other groups expected toundergo nucleophilic displacement as well as ketones, aldehydes,thioesters, olefins, alpha-beta unsaturated carbonyl groups, carbonatesand other electrophilic groups expected to undergo addition bynucleophiles.

In a preferred embodiment, A is an aliphatic linker of between 1-10carbon atoms or a substituted aryl ring of between 6-14 carbon atoms. Xis a functional group which does not react with azide groups and L is asuitable leaving group

In a second method for preparation of the acetylene-containing polymerderivatives of the invention, a PEG polymer having an average molecularweight from about 800 Da to about 100,000 Da, bearing either a protectedfunctional group or a capping agent at one terminus and a suitableleaving group at the other terminus is contacted by an acetylene anion.

An exemplary reaction scheme is shown below:

X-PEG-L+—C≡CR′→X-PEG-C≡CR′

wherein:

PEG is poly(ethylene glycol) and X is a capping group such as alkoxy ora functional group as described above; and

R′ is either H, an alkyl, alkoxy, aryl or aryloxy group or a substitutedalkyl, alkoxyl, aryl or aryloxy group.

In the example above, the leaving group L should be sufficientlyreactive to undergo SN2-type displacement when contacted with asufficient concentration of the acetylene anion. The reaction conditionsrequired to accomplish SN2 displacement of leaving groups by acetyleneanions are well known in the art.

Purification of the crude product can usually be accomplished byprecipitation of the product followed by chromatography, if necessary.

EXAMPLES

The following examples are offered to illustrate, but not to limit thepresent invention.

Example 1

The polyalkylene glycol (P—OH) is reacted with the alkyl halide (A) toform the ether (B). In these compounds, n is an integer from one to nineand R′ can be a straight- or branched-chain, saturated or unsaturatedC1, to C20 alkyl or heteroalkyl group. R′ can also be a C3 to C7saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, asubstituted or unsubstituted aryl or heteroaryl group, or a substitutedor unsubstituted alkaryl (the alkyl is a C1 to C20 saturated orunsaturated alkyl) or heteroalkaryl group. Typically, P—OH ispolyethylene glycol (PEG) or monomethoxy polyethylene glycol (mPEG)having a molecular weight of 800 to 40,000 Daltons (Da).

Example 2

mPEG-OH+Br—CH₂—CH≡CH→mPEG-O—CH₂—C≡CH

mPEG-OH with a molecular weight of 20,000 Da (mPEG-OH 20 kDa; 2.0 g, 0.1mmol, Sunbio) was treated with NaH (12 mg, 0.5 mmol) in THF (35 mL). Asolution of propargyl bromide, dissolved as an 80% weight solution inxylene (0.56 mL, 5 mmol, 50 equiv., Aldrich), and a catalytic amount ofKI were then added to the solution and the resulting mixture was heatedto reflux for 2 h. Water (1 mL) was then added and the solvent wasremoved under vacuum. To the residue was added CH₂Cl₂ (25 mL) and theorganic layer was separated, dried over anhydrous Na₂SO₄, and the volumewas reduced to approximately 2 mL. This CH₂Cl₂ solution was added todiethyl ether (150 mL) drop-wise. The resulting precipitate wascollected, washed with several portions of cold diethyl ether, and driedto afford propargyl-O-PEG.

Example 3

mPEG-OH+Br—(CH₂)₃—C≡CH→mPEG-O—(CH₂)₃—C≡CH

mPEG-OH with a molecular weight of 20,000 Da (mPEG-OH 20 kDa; 2.0 g, 0.1mmol, Sunbio) was treated with NaH (12 mg, 0.5 mmol) in THF (35 mL).Fifty equivalents of 5-chloro-1-pentyne (0.53 mL, 5 mmol, Aldrich) and acatalytic amount of KI were then added to the mixture. The resultingmixture was heated to reflux for 16 h. Water (1 mL) was then added andthe solvent was removed under vacuum. To the residue was added CH₂Cl₂(25 mL) and the organic layer was separated, dried over anhydrousNa₂SO₄, and the volume was reduced to approximately 2 mL. This CH₂Cl₂solution was added to diethyl ether (150 mL) drop-wise. The resultingprecipitate was collected, washed with several portions of cold diethylether, and dried to afford the corresponding alkyne.

Example 4

m-HOCH₂C₆H₄OH+NaOH+Br—CH₂—C≡CH→m-HOCH₂C₆H₄O—CH₂—C≡CH  (1)

m-HOCH₂C₆H₄O—CH₂—C≡CH+MsCl+N(Et)₃ →m-MsOCH₂C₆H₄O—CH₂—C≡CH  (2)

m-MsOCH₂C₆H₄O—CH₂—C≡CH+LiBr→m-Br—CH₂C₆H₄O—CH₂—C≡CH  (3)

mPEG-OH+m-Br—CH₂C₆H₄O—CH₂—C≡CH→mPEG-O—CH₂—C₆H₄O—CH₂—C≡CH  (4)

To a solution of 3-hydroxybenzylalcohol (2.4 g, 20 mmol) in THF (50 mL)and water (2.5 mL) was first added powdered sodium hydroxide (1.5 g,37.5 mmol) and then a solution of propargyl bromide, dissolved as an 80%weight solution in xylene (3.36 mL, 30 mmol). The reaction mixture washeated at reflux for 6 h. To the mixture was added 10% citric acid (2.5mL) and the solvent was removed under vacuum. The residue was extractedwith ethyl acetate (3×15 mL) and the combined organic layers were washedwith saturated NaCl solution (10 mL), dried over MgSO4 and concentratedto give the 3-propargyloxybenzyl alcohol.

Methanesulfonyl chloride (2.5 g, 15.7 mmol) and triethylamine (2.8 mL,20 mmol) were added to a solution of compound 3 (2.0 g, 11.0 mmol) inCH₂Cl₂ at 0° C. and the reaction was placed in the refrigerator for 16h. A usual work-up afforded the mesylate as a pale yellow oil. This oil(2.4 g, 9.2 mmol) was dissolved in THF (20 mL) and LiBr (2.0 g, 23.0mmol) was added. The reaction mixture was heated to reflux for 1 h andwas then cooled to room temperature. To the mixture was added water (2.5mL) and the solvent was removed under vacuum. The residue was extractedwith ethyl acetate (3×15 mL) and the combined organic layers were washedwith saturated NaCl solution (10 mL), dried over anhydrous Na₂SO₄, andconcentrated to give the desired bromide.

mPEG-OH 20 kDa (1.0 g, 0.05 mmol, Sunbio) was dissolved in THF (20 mL)and the solution was cooled in an ice bath. NaH (6 mg, 0.25 mmol) wasadded with vigorous stirring over a period of several minutes followedby addition of the bromide obtained from above (2.55 g, 11.4 mmol) and acatalytic amount of KI. The cooling bath was removed and the resultingmixture was heated to reflux for 12 h. Water (1.0) was added to themixture and the solvent was removed under vacuum. To the residue wasadded CH₂Cl₂ (25 mL) and the organic layer was separated, dried overanhydrous Na₂SO₄, and the volume was reduced to approximately 2 mL.Dropwise addition to an ether solution (150 mL) resulted in a whiteprecipitate, which was collected to yield the PEG derivative.

Example 5

mPEG-NH₂+X—C(O)—(CH₂)_(n)—C≡CR′→mPEG-NH—C(O)—(CH₂)_(n)—C≡CR′

The terminal alkyne-containing poly(ethylene glycol) polymers can alsobe obtained by coupling a poly(ethylene glycol) polymer containing aterminal functional group to a reactive molecule containing the alkynefunctionality as shown above.

Example 6

HO₂C—(CH₂)₂—C≡CH+NHS+DCC→NHSO—C(O)—(CH₂)₂—C≡CH  (1)

mPEG-NH₂+NHSO—C(O)—(CH₂)₂—C≡CH→mPEG-NH—C(O)—(CH₂)₂—C≡CH  (2)

4-pentynoic acid (2.943 g, 3.0 mmol) was dissolved in CH2Cl2 (25 mL).N-hydroxysuccinimide (3.80 g, 3.3 mmol) and DCC (4.66 g, 3.0 mmol) wereadded and the solution was stirred overnight at room temperature. Theresulting crude NHS ester 7 was used in the following reaction withoutfurther purification.

mPEG-NH₂ with a molecular weight of 5,000 Da (mPEG-NH₂, 1 g, Sunbio) wasdissolved in THF (50 mL) and the mixture was cooled to 4° C. NHS ester 7(400 mg, 0.4 mmol) was added portion-wise with vigorous stirring. Themixture was allowed to stir for 3 h while warming to room temperature.Water (2 mL) was then added and the solvent was removed under vacuum. Tothe residue was added CH₂Cl₂ (50 mL) and the organic layer wasseparated, dried over anhydrous Na₂SO₄, and the volume was reduced toapproximately 2 mL. This CH₂Cl₂ solution was added to ether (150 mL)drop-wise. The resulting precipitate was collected and dried in vacuo.

Example 7

This Example represents the preparation of the methane sulfonyl ester ofpoly(ethylene glycol), which can also be referred to as themethanesulfonate or mesylate of poly(ethylene glycol). The correspondingtosylate and the halides can be prepared by similar procedures.

mPEG-OH+CH₃SO₂Cl+N(Et)₃ →mPEG-O—SO₂CH₃ →mPEG-N₃

mPEG-OH (MW=3,400, 25 g, 10 mmol) in 150 mL of toluene wasazeotropically distilled for 2 hours under nitrogen and the solution wascooled to room temperature. To the solution was added 40 mL of dryCH₂Cl₂ and 2.1 mL of dry triethylamine (15 mmol). The solution wascooled in an ice bath and 1.2 mL of distilled methanesulfonyl chloride(15 mmol) was added dropwise. The solution was stirred at roomtemperature under nitrogen overnight and the reaction was quenched byadding 2 mL of absolute ethanol. The mixture was evaporated under vacuumto remove solvents, primarily those other than toluene, filtered,concentrated again under vacuum, and then precipitated into 100 mL ofdiethyl ether. The filtrate was washed with several portions of colddiethyl ether and dried in vacuo to afford the mesylate.

The mesylate (20 g, 8 mmol) was dissolved in 75 ml of THF and thesolution was cooled to 4° C. To the cooled solution was added sodiumazide (1.56 g, 24 mmol). The reaction was heated to reflux undernitrogen for 2 h. The solvents were then evaporated and the residuediluted with CH₂Cl₂ (50 mL). The organic fraction was washed with NaClsolution and dried over anhydrous MgSO₄. The volume was reduced to 20 mland the product was precipitated by addition to 150 ml of cold dryether.

Example 8

N₃—C₆H₄—CO₂H→N₃—C₆H₄CH₂OH  (1)

N₃—C₆H₄CH₂OH→Br—CH₂—C₆H₄—N₃  (2)

mPEG-OH+Br—CH₂—C₆H₄—N₃ →mPEG-O—CH₂—C₆H₄—N₃  (3)

4-azidobenzyl alcohol can be produced using the method described in U.S.Pat. No. 5,998,595. Methanesulfonyl chloride (2.5 g, 15.7 mmol) andtriethylamine (2.8 mL, 20 mmol) were added to a solution of4-azidobenzyl alcohol (1.75 g, 11.0 mmol) in CH₂Cl₂ at 0° C. and thereaction was placed in the refrigerator for 16 h. A usual work-upafforded the mesylate as a pale yellow oil. This oil (9.2 mmol) wasdissolved in THF (20 mL) and LiBr (2.0 g, 23.0 mmol) was added. Thereaction mixture was heated to reflux for 1 h and was then cooled toroom temperature. To the mixture was added water (2.5 mL) and thesolvent was removed under vacuum. The residue was extracted with ethylacetate (3×15 mL) and the combined organic layers were washed withsaturated NaCl solution (10 mL), dried over anhydrous Na₂SO₄, andconcentrated to give the desired bromide.

mPEG-OH 20 kDa (2.0 g, 0.1 mmol, Sunbio) was treated with NaH (12 mg,0.5 mmol) in THF (35 mL) and the bromide (3.32 g, 15 mmol) was added tothe mixture along with a catalytic amount of KI. The resulting mixturewas heated to reflux for 12 h. Water (1.0) was added to the mixture andthe solvent was removed under vacuum. To the residue was added CH₂Cl₂(25 mL) and the organic layer was separated, dried over anhydrousNa₂SO₄, and the volume was reduced to approximately 2 mL. Dropwiseaddition to an ether solution (150 mL) resulted in a precipitate, whichwas collected to yield mPEG-O—CH₂—C₆H₄—N₃.

Example 9

NH₂-PEG-O—CH₂CH₂CO₂H+N₃—CH₂CH₂CO₂—NHS→N₃—CH₂CH₂—C(O)NH-PEG-O—CH₂CH₂CO₂H

NH₂-PEG-O—CH₂CH₂CO₂H (MW 3,400 Da, 2.0 g) was dissolved in a saturatedaqueous solution of NaHCO₃ (10 mL) and the solution was cooled to 0° C.3-azido-1-N-hydroxysuccinimdo propionate (5 equiv.) was added withvigorous stirring. After 3 hours, 20 mL of H₂O was added and the mixturewas stirred for an additional 45 minutes at room temperature. The pH wasadjusted to 3 with 0.5 NH₂SO₄ and NaCl was added to a concentration ofapproximately 15 wt %. The reaction mixture was extracted with CH₂Cl₂(100 mL×3), dried over Na₂SO₄ and concentrated. After precipitation withcold diethyl ether, the product was collected by filtration and driedunder vacuum to yield the omega-carboxy-azide PEG derivative.

Example 10

mPEG-OMs+HC≡CLi→mPEG-O—CH₂—CH₂—C≡C—H

To a solution of lithium acetylide (4 equiv.), prepared as known in theart and cooled to −78 C in THF, is added dropwise a solution of mPEG-OMsdissolved in THF with vigorous stirring. After 3 hours, the reaction ispermitted to warm to room temperature and quenched with the addition of1 mL of butanol. 20 mL of H₂O is then added and the mixture was stirredfor an additional 45 minutes at room temperature. The pH was adjusted to3 with 0.5 NH₂SO₄ and NaCl was added to a concentration of approximately15 wt %. The reaction mixture was extracted with CH₂Cl₂ (100 mL×3),dried over Na₂SO₄ and concentrated. After precipitation with colddiethyl ether, the product was collected by filtration and dried undervacuum to yield the omega-carboxy-azide PEG derivative.

Example 11

The azide- and acetylene-containing amino acids were incorporatedsite-selectively into proteins using the methods described in L. Wang,et al., (2001), Science 292:498-500, J. W. Chin et al., Science301:964-7 (2003)), J. W. Chin et al., (2002), Journal of the AmericanChemical Society 124:9026-9027; J. W. Chin, & P. G. Schultz, (2002),Chem Bio Chem 11:1135-1137; J. W. Chin, et al., (2002), PNAS UnitedStates of America 99:11020-11024: and, L. Wang, & P. G. Schultz, (2002),Chem. Comm., 1-10. Once the amino acids were incorporated, thecycloaddition react was carried out with 0.01 mM protein in phosphatebuffer (PB), pH 8, in the presence of 2 mM PEG derivative, 1 mM CuSO₄,and ˜1 mg Cu-wire for 4 hours at 37° C.

Many modifications and other embodiments of the invention will come tomind to one skilled in the art to which this invention pertains havingthe benefit of teachings presented in the foregoing descriptions and theassociated drawings. Therefore, it is to be understood that theinvention is not to be limited to the specific embodiments disclosed andthat modifications and other embodiments are intended to be includedwithin the scope of the appended claims. Although specific terms areemployed herein, they are used in a generic and descriptive sense onlyand not for purposes of limitation. All publications, patents, andpatent applications cited herein are hereby incorporated by referencefor all purposes.

1-47. (canceled)
 48. A water-soluble compound comprising a polymer and at least one acetylene moiety, wherein said polymer is selected from the group consisting of poly(alkylene oxides), poly(oxyethylated polyols), and poly(olefinic alcohols), and wherein said polymer comprises at least one arm of a branched molecular structure.
 49. The water-soluble compound of claim 48 wherein said branched molecular structure is dendritic. 50-74. (canceled) 